The overall goal of this proposal is to provide postdoctoral training for Dr. Giordano da Silva at the interface of biophysics and biochemistry under the mentorship of Dr. Graham Palmer (Rice University). The research aims of the current project focus on biophysical characterization of a homolog of cyt b561, the CYTB561D2 gene product that has been dubbed "TScytb" because of its tumor suppressing activity. Biophysical characterization of TScytb is at an early and exciting stage, with many key structural and functional aspects remaining to be established, and the link between the redox behavior of the cytochrome and its anti-tumor activity yet to be explored. The proposed studies are designed to leverage the experience and methodology developed with cyt b561 to address some key issues regarding TScytb. Purified recombinant wild type and mutant TScytb proteins and cyt b561 chimeras will be used to accomplish the following specific aims: Aim 1: Characterize the axial ligation and ascorbate reaction kinetics of the hemes. The approach involves physico-chemical characterization of recombinant TScytb expressed in insect and yeast cell systems;Aim 2: Evaluate the roles of semidehydroascorbate and O2 as oxidants for TScytb. The approach involves kinetic analyses of the reactions of TScytb with SDA (generated by pulse radiolysis) and with oxygen;Aim 3: Determine which extramembrane segments of TScytb contribute to reaction with reductant and oxidant, and to tumor-suppressor activity. The approach involves characterization of the ascorbate reactivity and tumor suppression activity of cyt b561 chimeras bearing extramembrane domain substitutions from TScytb. This traineeship proposal combines a young researcher with an experienced and successful mentor, an excellent biomedical research environment, and an exciting research plan. The training program will promote Dr. da Silva's development as an independent researcher. The resulting structure/function information should make a fundamental contribution to understanding the relationship between the redox behavior of TScytb and its anti-tumor activity, understanding that may be useful in development of novel therapeutic strategies. PUBLIC HEALTH RELEVANCE: According to the Centers for Disease Control and Prevention, more people die of lung cancer than any other form of cancer, with approximately 200,000 people diagnosed and 160,000 deaths in 2005 alone [1];breast cancer claimed the lives of over 40,000 women in the same year [2]. Recently it was shown that one putative tumor suppressor in lung and breast cancers, the CYB561D2 gene product (also known as the101F6 protein or TScytb), belongs to the cytochrome b561 family [3]. The mechanism of tumor suppression is thought to involve ascorbate-dependent transmembrane electron transfer by Tscytb [3]. The proposed fellowship studies on biophysical characterization of TScytb and its interaction with ascorbate are designed to relate the cytochrome structure of TScytb with its anti-tumor activity;the results should aid the development of novel therapeutic strategies for cancer prevention and intervention. 1. ://www.cdc.gov/cancer/lung/ 2. ://www.cdc.gov/cancer/breast/ 3. Ohtani, S., Iwamaru, A., Deng, W., Ueda, K., Wu, G., Jayachandran, G., Kondo, S., Atkinson, E.N., Minna, J.D., Roth, J.A., Ji, L. Tumor suppressor 101F6 and ascorbate synergistically and selectively inhibit non-small cell lung cancer growth by caspase-independent apoptosis and autophagy. Cancer Res., 2007, 67, 6293-6303.